氯化铁诱导的动脉血栓形成和用于3D电子显微镜分析的样本采集
1Department of Molecular and Cellular Biochemistry,University of Kentucky, 2Department of Physiology and Cell Biology,University of Arkansas for Medical Sciences, 3Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering,National Institutes of Health
Summary
本协议描述了如何使用FeCl3介导的损伤来诱导动脉血栓形成，以及如何在血栓形成的各个阶段收集和制备动脉损伤样品以进行电子显微镜分析。
Abstract
心血管疾病是全世界死亡和发病的主要原因。异常血栓形成是糖尿病和肥胖等全身性疾病以及动脉粥样硬化、癌症和自身免疫性疾病等慢性炎症性疾病的共同特征。血管损伤时，凝血系统、血小板和内皮通常以协调的方式发挥作用，通过在损伤部位形成凝块来防止出血。这一过程中的异常导致出血过多或血栓形成不受控制/抗血栓形成活性不足，这转化为血管闭塞及其后遗症。FeCl3诱导的颈动脉损伤模型是探索血栓形成如何在 体内启动和进展的宝贵工具。该模型涉及内皮损伤/剥落以及随后在受伤部位形成凝块。它提供了一种高度灵敏的定量测定，以监测不同程度的血管损伤和凝块形成。一旦优化，这种标准技术可用于研究血栓形成的分子机制，以及生长中的血栓中血小板的超微结构变化。该测定也可用于研究抗血栓形成和抗血小板药物的疗效。本文解释了如何启动和监测FeCl3诱导的动脉血栓形成，以及如何收集样品通过电子显微镜进行分析。
Introduction
血栓形成是部分或完全阻塞血管的血凝块的形成，阻碍血液的自然流动。这会导致严重和致命的心血管事件，如缺血性心脏病和中风。心血管疾病是发病和死亡的主要原因，导致全世界四分之一的死亡1，2，3。虽然血栓形成表现为血管系统功能障碍，但它可能是潜在的微生物或病毒感染、免疫紊乱、恶性肿瘤或代谢疾病的结果。血液流动由血管系统不同组成部分（包括内皮细胞、红/白细胞、血小板和凝血因子）之间的复杂相互作用维持4.血管损伤后，血小板与内皮下基质上的粘附蛋白相互作用并释放其颗粒内容物，从而募集更多的血小板5。同时，凝血级联反应被激活，导致纤维蛋白形成和沉积。最终，形成凝块，包含被困在纤维蛋白网内的血小板和红细胞6。虽然抗血小板和抗凝药物可用于调节血栓形成，但假性出血仍然是这些疗法的主要问题，需要微调这些药物的剂量和组合。因此，仍然迫切需要发现新的抗血栓药物7。
使用多种方法研究血栓形成以造成血管损伤：机械（血管结扎），热损伤（激光损伤）和化学损伤（FeCl3 / Rose Bengal应用）。血栓形成的性质因损伤部位（动脉与静脉）、损伤方法或程度而异。在所有这些类型中，FeCl3诱导的血管损伤是最广泛使用的方法。它已被用于小鼠，大鼠，兔子，豚鼠和狗8，9，10，11，12。该方法相对简单，易于使用，如果主要参数标准化，则在各种血管系统（例如动脉[颈动脉和股动脉]，静脉[颈动脉]和小动脉[提睾和肠系膜]）中具有敏感性和可重复性（补充表1）。
该模型也可用于进一步了解凝块形成的机制和形态。该技术独特地提供了在不同流速点阻止血栓形成的优势，以在过程闭塞之前研究该过程的中间阶段。血栓形成研究的最新进展使用该模型将注意力集中在溶栓的非药物方法13 或抗血栓和/或纤维蛋白溶解剂的非侵入性递送14，15上。一些研究小组已经表明，当血小板膜涂有这些疗法时，药物可以在热刺激下被激活以靶向凝块16。这里描述的技术可用于在单个血小板水平验证其发现等研究。在本手稿中，方案 1 描述了基本的 FeCl3 介导的血管损伤程序，而方案 2 描述了收集和固定血管损伤样本以通过电子显微镜进一步分析的方法。
Protocol
这里讨论的所有实验都经过肯塔基大学机构动物护理和使用委员会（IACUC）的审查和批准。 注：手术器械列于 图1 和 材料表中。使用C57BL / 6J小鼠，8-10周龄，雄性/雌性或相关的遗传操作（敲除或敲入）菌株。 1. 氯化铁3诱发的颈动脉损伤 小鼠麻醉诱导称量鼠标。 通过腹膜内（i…
Representative Results
数据通常表示为闭塞时间，或形成完全闭塞血栓所需的时间。这些数据可以绘制为Kaplan-Meier生存曲线（图4A）19，带有条形图的点图，显示血流停止或实验终止时的最终血流（图4B），或折线图（图4C）。可以使用这种技术研究血栓稳定性。在大多数情况下，在FeCl3损伤时，血栓逐渐形成，随着其生长，血?…
Discussion
将FeCl3局部应用于脉管系统以诱导血栓形成是一种广泛使用的技术，并且有助于建立各种血小板受体，配体信号通路及其抑制剂的作用20，21，22，23。FeCl3引起血栓形成的机制是多方面的;以前，内皮剥蚀被认为是血栓形成的原因，然而近年来，多份报告表明红细胞和血浆蛋白在此过程?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢白心实验室的成员仔细阅读这份手稿。这项工作得到了NIH，NHLBI（HL56652，HL138179和HL150818）的资助，以及退伍军人事务部对SWW的优异奖，R01 HL 155519到BS，以及NIBIB对R.D.L.的校内计划资助。
Materials
| 0.9% Saline | Fisher Scientific | BP358-212 | NaCl used to make a solution of 0.9% saline |
| 1 mL Syringe | Becton, Dickinson and Company | 309659 | |
| 190 Proof Ethanol | KOPTEC | V1101 | Used to make a 70% ethanol solution to use for prepping the mouse for surgery |
| 2,2,2 Tribromoethanol | Sigma Aldrich | 48402 | |
| 25 Yard Black Braided Silk Suture (5-0) | DEKNATEL | 136082-1204 | |
| 26G x 3/8 Needle | Becton, Dickinson and Company | 305110 | |
| 2-methyl-2-butanol | Sigma Aldrich | 240486 | |
| 7.5 mL Transfer Pipet, Graduated to 3 mL | Globe Scientific Inc. | 135010 | |
| Alcohol Prep Pads (70% Isopropyl Alcohol) | Medline | MDS090735 | |
| Araldite GY 502 | Electron microscopy Services | 10900 | |
| Cell Culture Dish 35mm X 10mm | Corning Incorporated | 430165 | |
| Compact Scale | Ward's Science | 470314-390 | |
| Dissecting Scissors, 12.5 cm long | World Precision Instrument | 15922-G | |
| DMP-30 activator | Electron microscopy Services | 13600 | |
| Dodenyl Succinic Anhydride/ DDSA | Electron microscopy Services | 13700 | |
| Doggy Poo Bags/animal carcass disposal bag | Crown Products | PP-RB-200 | |
| Doppler FlowProbe | Transonic Systems Inc. | MA0.5PSB | |
| EMBED 812 resin | Electron microscopy Services | 14900 | |
| Ethyl Alcohol, anhydrous 200 proof | Electron microscopy Services | 15055 | |
| Eye Dressing Forceps, 4" Full Curved, Standard, 0.8mm Wide Tips | Integra Miltex | 18-784 | |
| Filter Paper | VWR | 28310-106 | |
| Fine Scissors – Sharp-Blunt | Fine Science Tools | 14028-10 | |
| Finger Loop Ear Punches | Fine Science Tools | 24212-01 | |
| Gauze Sponges 2” x 2” – 12 Ply | Dukal Corporation | 2128 | |
| Glutaraldehyde (10% solution) | Electron microscopy Services | 16120 | |
| Integra Miltex Carbon Steel Surgical Blade #10 | Integra® Miltex® | 4110 | |
| Iron (III) Chloride | SIGMA-ALDRICH | 157740-100G | |
| Knife Handle Miltex® Extra Fine Stainless Steel Size 3 | Integra Lifesciences | 157510 | |
| L-aspartic acid | Sigma Fisher | A93100 | |
| L-aspartic acid | Fisher Scientific | BP374-100 | |
| Lead Nitrate | Fisher Scientific | L-62 | |
| LEICA S8AP0 Microscope | LEICA | No longer available | No longer available from the company |
| LEICA S8AP0 Microscope Stand | LEICA | 10447255 | No longer available from the company |
| Light-Duty Tissue Wipers | VWR | 82003-822 | |
| Micro Dissecting Forceps; 1×2 Teeth, Full Curve; 0.8 mm Tip Width; 4" Length | Roboz Surgical Instrument Company | RS-5157 | |
| Osmium Tetroxide 4% aqueous solution | Electron microscopy Services | 19150 | |
| Paraformaldehyde (16% solution) | Electron microscopy Services | 15710 | |
| Potassium ferricyanide | SIGMA-ALDRICH | P-8131 | |
| Propylene Oxide, ACS reagent | Electron microscopy Services | 20401 | |
| Rainin Classic Pipette PR-10 | Rainin | 17008649 | |
| Research Flowmeter | Transonic Systems Inc. | T402B01481 | Model: T402 |
| Scotch Magic Invisible Tape, 3/4" x 1000", Clear | Scotch | 305289 | |
| Small Animal Heated Pad | K&H Manufacturing Inc. | Model: HM10 | |
| Sodium Cacodylate Buffer 0.2M, pH7.4 | Electron microscopy Services | 11623 | |
| Sterile Cotton Tipped Applicators | Puritan Medical Products | 25-806 1WC | |
| Steromaster Illuminator | Fisher Scientific | 12-562-21 | No longer available from the company |
| Surgical Dumont #7 Forceps | Fine Science Tools | 11271-30 | |
| Thiocarbohydrazide (TCH) | SIGMA-ALDRICH | 88535 | |
| Universal Low Retention Pipet Tip Reloads (0.1-10 µL) | VWR | 76323-394 | |
| Uranyl Acetate | Electron microscopy Services | 22400 | |
| Veet Gel Cream Hair Remover | Reckitt Benckiser | 3116875 | |
| White Antistatic Hexagonal Weigh Boats, Medium, 64 x 15 x 19 mm | Fisher Scientific | S38975 | |
| WinDAQ/100 Software for Windows | DATAQ Instruments, Inc. | Version 3.38 | Freely available to download. https://www.dataq.com/products/windaq/ |
| ZEISS AxioCam Icc 1 | ZEISS | 57615 |
References
- Lozano, R., et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 380 (9859), 2095-2128 (2012).
- Raskob, G. E., et al. Thrombosis: a major contributor to global disease burden. Arteriosclerosis, Thrombosis, and Vascular Biology. 34 (11), 2363-2371 (2014).
- Walton, J. Lead aspartate, an en bloc contrast stain particularly useful for ultrastructural enzymology. Journal of Histochemistry and Cytochemistry. 27 (10), 1337-1342 (1979).
- Palta, S., Saroa, R., Palta, A. Overview of the coagulation system. Indian Journal of Anaesthesia. 58 (5), 515-523 (2014).
- Joshi, S., Whiteheart, S. W. The nuts and bolts of the platelet release reaction. Platelets. 28 (2), 129-137 (2017).
- Periayah, M. H., Halim, A. S., Mat Saad, A. Z. Mechanism action of platelets and crucial blood coagulation pathways in hemostasis. International Journal of Hematology-Oncology and Stem Cell Research. 11 (4), 319-327 (2017).
- Alexopoulos, D., Katogiannis, K., Sfantou, D., Lekakis, J. Combination antiplatelet treatment in coronary artery disease patients: A necessary evil or an overzealous practice. Platelets. 29 (3), 228-237 (2018).
- Kurz, K. D., Main, B. W., Sandusky, G. E. Rat model of arterial thrombosis induced by ferric chloride. Thrombosis Research. 60 (4), 269-280 (1990).
- Denis, C. V., et al. Towards standardization of in vivo thrombosis studies in mice. Journal of Thrombosis and Haemostasis. 9 (8), 1641-1644 (2011).
- Marsh Lyle, E., et al. Assessment of thrombin inhibitor efficacy in a novel rabbit model of simultaneous arterial and venous thrombosis. Thrombosis and Haemostasis. 79 (3), 656-662 (1998).
- Kato, Y., et al. Inhibition of arterial thrombosis by a protease-activated receptor 1 antagonist, FR171113, in the guinea pig. European Journal of Pharmacology. 473 (2-3), 163-169 (2003).
- Huttinger, A. L., et al. Ferric chloride-induced canine carotid artery thrombosis: a large animal model of vascular injury. Journal of Visualized Experiments. (139), e57981 (2018).
- Zhang, W., et al. Antithrombotic therapy by regulating the ROS-mediated thrombosis microenvironment and specific nonpharmaceutical thrombolysis Using Prussian blue nanodroplets. Small. 18 (15), 2106252 (2022).
- Liu, B., et al. Platelet membrane cloaked nanotubes to accelerate thrombolysis by thrombus clot-targeting and penetration. Small. , 2205260 (2022).
- Refaat, A., et al. Near-infrared light-responsive liposomes for protein delivery: Towards bleeding-free photothermally-assisted thrombolysis. Journal of Controlled Release. 337, 212-223 (2021).
- Li, S., et al. Biomimetic nanoplatelets to target delivery hirudin for site-specific photothermal/photodynamic thrombolysis and preventing venous thrombus formation. Small. 18 (51), 2203184 (2022).
- Subramaniam, S., Kanse, S. M. Ferric chloride-induced arterial thrombosis in mice. Current Protocols in Mouse Biology. 4 (4), 151-164 (2014).
- Cocchiaro, J. L., Kumar, Y., Fischer, E. R., Hackstadt, T., Valdivia, R. H. Cytoplasmic lipid droplets are translocated into the lumen of the Chlamydia trachomatis parasitophorous vacuole. Proceedings of the National Academy of Sciences. 105 (27), 9379-9384 (2008).
- Kaplan, E. L., Meier, P. Nonparametric-estimation from incomplete observations. Journal of the American Statistical Association. 53 (282), 457-481 (1958).
- Chauhan, A. K., Kisucka, J., Lamb, C. B., Bergmeier, W., Wagner, D. D. von Willebrand factor and factor VIII are independently required to form stable occlusive thrombi in injured veins. Blood. 109 (6), 2424-2429 (2007).
- Andre, P., et al. CD40L stabilizes arterial thrombi by a beta3 integrin–dependent mechanism. Nature Medicine. 8 (3), 247-252 (2002).
- Ni, H., et al. Persistence of platelet thrombus formation in arterioles of mice lacking both von Willebrand factor and fibrinogen. Journal of Clinical Investigation. 106 (3), 385-392 (2000).
- Bergmeier, W., et al. The role of platelet adhesion receptor GPIbalpha far exceeds that of its main ligand, von Willebrand factor, in arterial thrombosis. Proceedings of the National Academy of Sciences. 103 (45), 16900-16905 (2006).
- Ciciliano, J. C., et al. Resolving the multifaceted mechanisms of the ferric chloride thrombosis model using an interdisciplinary microfluidic approach. Blood. 126 (6), 817-824 (2015).
- Eckly, A., et al. Mechanisms underlying FeCl3-induced arterial thrombosis. Journal of Thrombosis and Haemostasis. 9 (4), 779-789 (2011).
- Woollard, K. J., Sturgeon, S., Chin-Dusting, J. P. F., Salem, H. H., Jackson, S. P. Erythrocyte hemolysis and hemoglobin oxidation promote ferric chloride-induced vascular injury. Journal of Biological Chemistry. 284 (19), 13110-13118 (2009).
- Shim, Y., et al. Characterization of ferric chloride-induced arterial thrombosis model of mice and the role of red blood cells in thrombosis acceleration. Yonsei Medical Journal. 62 (11), 1032-1041 (2021).
- Ghosh, S., et al. Evaluation of the prothrombotic potential of four-factor prothrombin complex concentrate (4F-PCC) in animal models. PLoS One. 16 (10), 0258192 (2021).
- Wilbs, J., et al. Cyclic peptide FXII inhibitor provides safe anticoagulation in a thrombosis model and in artificial lungs. Nature Communications. 11 (1), 3890 (2020).
- Wei, Y., Deng, X., Sheng, G., Guo, X. B. A rabbit model of cerebral venous sinus thrombosis established by ferric chloride and thrombin injection. Neuroscience Letters. 662, 205-212 (2018).
- Jacob-Ferreira, A. L., et al. Antithrombotic activity of Batroxase, a metalloprotease from Bothrops atrox venom, in a model of venous thrombosis. International Journal of Biological Macromolecules. 95, 263-267 (2017).
- Zhou, X., et al. A rabbit model of cerebral microembolic signals for translational research: preclinical validation for aspirin and clopidogrel. Journal of Thrombosis and Haemostasis. 14 (9), 1855-1866 (2016).
- Yang, X., et al. Effect of evodiamine on collagen-induced platelet activation and thrombosis. BioMed Research International. 2022, 4893859 (2022).
- Li, W., McIntyre, T. M., Silverstein, R. L. Ferric chloride-induced murine carotid arterial injury: A model of redox pathology. Redox Biology. 1 (1), 50-55 (2013).
- Li, W., Nieman, M., Sen Gupta, A. Ferric chloride-induced murine thrombosis models. Journal of Visualized Experiments. (115), e54479 (2016).
- Holly, S. P., et al. Ether lipid metabolism by AADACL1 regulates platelet function and thrombosis. Blood Advances. 3 (22), 3818-3828 (2019).
- Bird, J. E., et al. Prediction of the therapeutic index of marketed anti-coagulants and anti-platelet agents by guinea pig models of thrombosis and hemostasis. Thrombosis Research. 123 (1), 146-158 (2008).
Cite This Article
Joshi, S., Smith, A. N., Prakhya, K. S., Alfar, H. R., Lykins, J., Zhang, M., Pokrovskaya, I., Aronova, M., Leapman, R. D., Storrie, B., Whiteheart, S. W. Ferric Chloride-Induced Arterial Thrombosis and Sample Collection for 3D Electron Microscopy Analysis. J. Vis. Exp. (193), e64985, doi:10.3791/64985 (2023).